Wednesday, February 10, 2016

Although there are still a lot of unanswered questions, today's Early Release from the MMWR contains some of the strongest evidence to date linking maternal infection with the Zika virus to microcephaly and/or fetal death.

Analysed are four cases from Brazil, two involving fetuses that spontaneously miscarried late in the first trimester, and two babies born with microcephaly at or near full term that died within 20 hours of delivery.

All four mothers had clinical signs of Zika infection during their first trimester, and subsequent post mortem testing of brain and placental tissues (see details below), showed evidence of Zika infection.

FEBRUARY 10, 2016
A surge in the number of children born with microcephaly has been noted
in regions of Brazil with a high prevalence of suspected Zika virus
disease. This report describes evidence of a link between Zika virus
infection and microcephaly and fetal demise through detection of viral
RNA and antigens in brain tissues from infants with microcephaly and
placental tissues from early miscarriages.

Zika virus is a mosquito-borne flavivirus that is related to dengue virus and transmitted primarily by Aedes aegypti
mosquitoes, with humans acting as the principal amplifying host during
outbreaks. Zika virus was first reported in Brazil in May 2015 (1).
By February 9, 2016, local transmission of infection had been reported
in 26 countries or territories in the Americas.* Infection is usually
asymptomatic, and, when symptoms are present, typically results in mild
and self-limited illness with symptoms including fever, rash,
arthralgia, and conjunctivitis. However, a surge in the number of
children born with microcephaly was noted in regions of Brazil with a
high prevalence of suspected Zika virus disease cases. More than 4,700
suspected cases of microcephaly were reported from mid-2015 through
January 2016, although additional investigations might eventually result
in a revised lower number (2). In response, the Brazil Ministry
of Health established a task force to further investigate possible
connections between the virus and brain anomalies in infants (3).

Since November 2015, CDC has been developing assays for Zika virus
testing in formalin-fixed, paraffin-embedded (FFPE) tissue samples. In
December 2015, FFPE tissues samples from two newborns (born at 36 and 38
weeks gestation) with microcephaly who died within 20 hours of birth
and two miscarriages (fetal losses at 11 and 13 weeks) were submitted to
CDC, from the state of Rio Grande do Norte in Brazil, for
histopathologic evaluation and laboratory testing for suspected Zika
virus infection. All four mothers had clinical signs of Zika virus
infection, including fever and rash, during the first trimester of
pregnancy, but did not have clinical signs of active infection at the
time of delivery or miscarriage. The mothers were not tested for
antibodies to Zika virus. Samples included brain and other autopsy
tissues from the two newborns, a placenta from one of the newborns, and
products of conception from the two miscarriages.

FFPE tissues were tested by Zika virus reverse
transcription-polymerase chain reaction (RT-PCR) targeting the
nonstructural protein 5 and envelope genes using general methods for
RT-PCR (4), and by immunohistochemistry using a mouse polyclonal anti-Zika virus antibody, using methods previously described (5).
Specific specimens from all four cases were positive by RT-PCR, and
sequence analysis provided further evidence of Zika virus infection,
revealing highest identities with Zika virus strains isolated from
Brazil during 2015. In the newborns, only brain tissue was positive by
RT-PCR assays.

Specimens from two of the four cases were positive by
immunohistochemistry: viral antigen was noted in mononuclear cells
(presumed to be glial cells and neurons within the brain) of one
newborn, and within the chorionic villi from one of the miscarriages.
Testing for dengue virus was negative by RT-PCR in specimens from all
cases.

For both newborns, significant histopathologic changes were limited
to the brain, and included parenchymal calcification, microglial
nodules, gliosis, and cell degeneration and necrosis. Other autopsy
tissues and placenta had no significant findings. Tests for
toxoplasmosis, rubella, cytomegalovirus, herpes simplex, and HIV were
negative in the two mothers who experienced miscarriages. Placental
tissue from one miscarriage showed heterogeneous chorionic villi with
calcification, fibrosis, perivillous fibrin deposition, and patchy
intervillositis and focal villitis, while tissue from the other
miscarriage had sparsely sampled normal-appearing chorionic villi.

This report describes evidence of a link between Zika virus infection
and microcephaly and fetal demise through detection of viral RNA and
antigens in brain tissues from infants with microcephaly and placental
tissues from early miscarriages. Histopathologic findings indicate the
presence of Zika virus in fetal tissues. These findings also suggest
brain and early gestational placental tissue might be the preferred
tissues for postmortem viral diagnosis. Nonfrozen, formalin-fixed
specimens or FFPE blocks are the preferred sample type for
histopathologic evaluation and immunohistochemistry, and RT-PCR can be
performed on either fresh frozen or formalin-fixed specimens. To better
understand the pathogenesis of Zika virus infection and associated
congenital anomalies and fetal death, it is necessary to evaluate
autopsy and placental tissues from additional cases, and to determine
the effect of gestational age during maternal illness on fetal outcomes.